The principal goal of the proposed research is to study the biochemistry and genetics of replication of yeast chromosomal DNA. In particular, the work will focus on two projects: (1) elucidation of the mechanisms that contribute to the fidelity of DNA replication, and (2) determination of the in vivo replication fork. The yeast DNA polymerase I-exonuclease complex will be purified and the fidelity of DNA replication determined in in vitro assays. Using antibodies directed against the exonuclease subunit or a partial amino acid sequence of this subunit, its gene will be cloned. A mutational analysis of this gene will determine the importance of proofreading during DNA replication of this eucaryote. Other mechanisms that lead to a high overall fidelity of DNA duplication will also be investigated. The yeast replication fork will be studied in vivo using pulse labeling methods to determine whether DNA replication in yeast is discontinuous or semidiscontinuous. The proposed study of eukaryotic replication will focus on yeast because, of all eukaryotic organisms, yeast is the one most amenable to biochemical and genetic manipulation. An intensive study of DNA replication and its regulation in yeast will undoubtedly be of significance in understanding cell growth and division in higher organisms. In addition, a better understanding of the mechanisms that induce or prevent mutations in yeast may lead to a better understanding of similar mechanisms in humans.